Studies on the Membrane-Active Behavior of Outer Membrane Vesicles from a Gram Negative Bacterium

Abstract

Gram-negative bacteria are known to produce small ∼50-200 nm vesicles by “blebbing” of their outer membranes. These outer membrane vesicles (OMV) have been implicated in activities such as transmission of virulence factors, horizontal gene transfer and development of biofilms. In this investigation, the interactions of OMV from Lysobacter enzymogenes (strain C3) with other membranes have been monitored using fluorescent assays for association and/or fusion. Defined composition large unilamellar vesicles labeled with the fluorescence resonance energy transfer (FRET) pair 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) were incubated with isolated OMV to observe the interaction via the FRET ratio of donor/acceptor. OMV substantially increased this ratio over the course of about an hour (t1/2 ∼10-20 min.) at 30°C, when added directly to vesicles comprising disordered or fluid lipids, such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). This apparent fusion was much more limited or absent with liquid ordered membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (DPPC/chol). In the case of POPG/POPE 1/3 vesicles, apparent fusion was also confirmed by testing vesicles with exclusively inner monolayer fluorescent probes to diminish any fluorescence changes from external interactions of outer monolayer fluorophores. When the OMV were labeled with the fluorophore 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine, and incubated with a natural host, yeast cells (strain S30), association and/or fusion was observed by co-sedimentation of the OMV label with yeast cells. These studies may help to elucidate the mechanism of natural host-pathogen interactions and define the lipid specificity for the design of liposomes for fusogenic delivery to OMV targets. There is therapeutic interest in delivery to Gram-negative OMV, e.g. where they exist in biofilm infections, or to elucidate a semi-synthetic approach to modify OMV lipid composition to generate useful delivery vehicles.